Detecting photons and producing images from a scanned field of view have been performed to produce electronic outputs representing the field of view of an instrument, such as a laser-scanning confocal microscope (LSCM). In this regard, the term “photon” means a unit of electromagnetic energy irrespective of its position in the spectrum, e.g., visible or invisible radiation. In quantum physics, a photon is characterized as an entity having particle and wave characteristics. Other forms of radiation, such as electrons, may also exhibit both particle and wave characteristics. The nature of the present invention and the manner of its use are not dependent on whether the radiant events are photons or other types of elementary particles.
In one prior-art optical-detection technique, photons are directed by a confocal imager in a confocal microscope to be sensed by a detector. A confocal imager comprises a point-source of light that illuminates a spot on or in a specimen. In order to illuminate an entire specimen with the spot, the light source is scanned across the specimen by a beam-steering device using scanners that are well known in the art. An illuminated spot is imaged onto a detector through a pinhole. Detectors comprise, for example, avalanche photodiode arrays or photomultiplier tubes or arrays of such devices.
The light source, the illuminated spot, and the detector have the same foci and are placed in conjugate focal-planes. Hence, they are “confocal” to each other.
The diameter of the pinhole is preferably matched to the illuminated spot through the optics situated between them. Because a small spot is illuminated and detected through a small aperture, light imaged onto the detector comes predominantly from the plane in focus within or on the specimen. The detector produces output pulses indicative of the detected photons.
The output pulses from the detector are processed to provide information such as time-correlated photon-counting histograms and image-generation in conventional laser scanning. In conventional imaging systems, however, photons obtained over each of a number of successive, selected equal time periods defined by a pixel clock are used to generate a respective intensity value assigned to each pixel (a pixel is a two-dimensional area of a portion of an image). Photon counts are “binned,” that is, accumulated as groups, during each sampling period; each group corresponds to a pixel location of an image display. (It is noted that the term “binning” is sometimes used to denote lumping pixels together, e.g., as during use of a CCD camera. This is a different use of “binning” than the use of the term herein.) In this manner, a computer builds up an entire image one pixel at a time to produce a two-dimensional image often made up of multiple thousands or millions of pixels. For three-dimensional imaging, successive two-dimensional layers of a specimen are scanned, and the computer builds up an image comprising voxels (three-dimensional pixels).
In producing a conventional image, a scan rate is selected. As scan rate increases, fewer photons per pixel per scan are accumulated, and the intensity of the pixels and their signal-to-noise ratios therefore decrease. As a result, prior-art pixel-based imaging systems face constraints in scan rate with regard to the quality of output signal to be produced. Physical and mechanical constraints, such as the rate at which a scanner can move, are also present. In addition, the number of photon counts in a specimen affects other parameters of image quality relating to intensity. These parameters include signal-to-noise ratio.
As a result, pixel-based scanning typically allows for reduced flexibility in experiment design. Resolution of the location of each photon is limited to the dimensions of a pixel or voxel as applicable. The amount of excitation illumination required for the output data to reach convergence of features of sensed images is proportional to the number of photons that must be produced to provide data sufficient to reach this convergence. When pixels are of smaller dimension and therefore provide fewer photons per scan, specimens would have to be subjected to excitation radiation a larger number of times or the same number of times (but for longer time intervals) than if the pixels were larger.
The requirement for greater illumination has functional drawbacks. In example applications involving fluorescent specimens, many fluorescent molecules under test can fluoresce only a limited number of times. At some point, response to excitation radiation ceases, and an effect known as photo-bleaching occurs. Over-illumination also presents another drawback. With measurements made in vivo, emission of photons from tissue produces free radicals, which can damage cells. Therefore, over-illumination of tissue can result in photo-toxicity.
A limitation of typical prior-art techniques is that they are optically based. Optically based techniques have an inherent limit of resolution known as a diffraction limit, which may be ˜0.6λ, where λ is the wavelength of the illuminating light. The resolving power of a lens is ultimately limited by diffraction effects. The lens's aperture is a “hole” that is analogous to a two-dimensional version of the single-slit experiment. Light passing through the lens interferes with itself, creating a ring-shaped diffraction pattern known as the Airy pattern, that blurs the image. An empirical diffraction limit is given by the Rayleigh criterion:
            sin      ⁢                          ⁢      θ        =          1.22      ⁢              λ        D              ,where θ is the angular resolution, λ is the wavelength of light, and D is the diameter of the lens. A wave does not have to pass through an aperture to diffract. For example, a beam of light of a finite size passing through a lens also undergoes diffraction and spreads in diameter. This effect limits the minimum size d of spot of light formed at the focal point of a lens, known as the diffraction limit:
      d    =          2.44      ⁢      λ      ⁢              f        a              ,where λ is the wavelength of the light, f is the focal length of the lens, and a is the diameter of the beam of light, or (if the beam is filling the lens) the diameter of the lens. Techniques that utilize so-called far-field or propagating-wave optics do not afford the opportunity to obtain resolution beyond the diffraction limit.